Cell Death and Differentiation (2010) 17, 1189–1198 & 2010 Macmillan Publishers Limited All rights reserved 1350-9047/10 $32.00 www.nature.com/cdd Cyclin-C-dependent cell-cycle entry is required for activation of non-homologous end joining DNA repair in postmitotic neurons A Tomashevski1, DR Webster2, P Grammas1,3, M Gorospe4 and II Kruman*,1,3 It is commonly believed that neurons remain in G0 phase of the cell cycle indefinitely. Cell-cycle re-entry, however, is known to contribute to neuronal apoptosis. Moreover, recent evidence demonstrates the expression of cell-cycle proteins in differentiated neurons under physiological conditions. The functional roles of such expression remain unclear. Since DNA repair is generally attenuated by differentiation in most cell types, the cell-cycle-associated events in postmitotic cells may reflect the need to re-enter the cell cycle to activate DNA repair. We show that cyclin-C-directed, pRb-dependent G0 exit activates the non- homologous end joining pathway of DNA repair (NHEJ) in postmitotic neurons. Using RNA interference, we found that abrogation of cyclin-C-mediated exit from G0 compromised DNA repair but did not initiate apoptosis. Forced G1 entry combined with prevention of G1-S progression triggered NHEJ activation even in the absence of DNA lesions, but did not induce apoptosis in contrast to unrestricted progression through G1-S. We conclude that G0-G1 transition is functionally significant for NHEJ repair in postmitotic neurons. These findings reveal the importance of cell-cycle activation for controlling both DNA repair and apoptosis in postmitotic neurons, and underline the particular role of G1-S progression in apoptotic signaling, providing new insights into the mechanisms of DNA damage response (DDR) in postmitotic neurons. Cell Death and Differentiation (2010) 17, 1189–1198; doi:10.1038/cdd.2009.221; published online 29 January 2010 Due to continuous exposure to genotoxic stress resulting from repairable DNA damage, also support the existence of a exogenous and endogenous sources, protection of genomic link between the cell-cycle machinery and DNA repair in integrity is a major challenge for living cells. Failure to repair postmitotic cells.12 DNA lesions often leads to cell death, genomic instability, and Although cells in most mammalian tissues enter a terminally 1,2 tumorigenesis. Terminally differentiated neurons are highly arrested (G0) phase at some point during their life, the susceptible to oxidative DNA damage due to their high rate of mechanisms regulating the maintenance of G0 and G0-G1 oxidative metabolism.3 For this reason, DNA repair is highly transition are not fully understood. The retinoblastoma protein important for these cells.2,4 The mechanisms of DNA repair (pRb) plays a crucial role in cell-cycle regulation.13 Recently, have been investigated mainly in proliferating cells. In these the cyclin-C/cyclin-dependent kinase-3 (CDK3) complex was cells, the cell-cycle machinery is a part of the DNA-damage shown to facilitate exit from G0 into the cell cycle by pRb response (DDR), involved in both DNA repair and apoptosis.5 phosphorylation at serines (S) 807 and 811. This cyclin-C- DNA repair in terminally differentiated cells is not expected to associated kinase activity peaked shortly after mitogenic 14 be linked to the cell cycle. However, increasing evidence stimulation of quiescent cells in early G1. During exit from indicates that the cell-cycle machinery plays a key role in G0, the cyclin-C/CDK3 complex directed pRb phosphorylation different processes in terminally differentiated cells;6,7 for at S807/811 in a temporal pattern that preceded pRb example, cell-cycle activation is essential for apoptotic phosphorylation by cyclin-D/CDK4, cyclin-D/CDK6, and signaling in neurons.3,8,9 Expression of cell-cycle proteins cyclin-E/CDK2.14 The levels of cyclin-E protein and asso- 15 has been observed at physiological conditions in terminally ciated kinase activity rise in the late G1 phase. differentiated neurons,7 but the functional relevance of this As double-strand breaks (DSBs) are the most lethal form expression remains unknown. Since DNA repair is generally of DNA damage,16 maintenance of genomic integrity attenuated by differentiation in most types of cells,10,11 the depends on efficient and accurate DSB repair. Mammalian expression of cell-cycle-related proteins may reflect the need cells repair DSBs by two pathways: homologous recombina- of resting cells to re-enter the cell cycle in order to activate tion and non-homologous end-joining (NHEJ). NHEJ is DNA repair. Our recent data demonstrating that cell-cycle- predominant in mammalian cells.17 NHEJ starts with the related proteins are expressed in neurons exposed to binding of Ku70/Ku80 (Ku) heterodimer to the broken DNA 1Garrison Institute on Aging, Texas Tech University Health Sciences Center, Lubbock, TX, USA; 2Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, USA; 3Department of Neurology, Texas Tech University Health Sciences Center, Lubbock, TX, USA and 4Laboratory of Cellular and Molecular Biology, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA *Corresponding author: II Kruman, Garrison Institute on Aging and Department of Neurology, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA. Tel: þ 1 806 743 2820; Fax: þ 1 806 743 2698; E-mail: [email protected] Keywords: neuron; DNA repair; cell cycle; DNA damage; apoptosis Abbreviations: CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; DNA–PKcs, DNA–protein-kinase catalytic subunit; DSB, double-strand break; NHEJ, non-homologous end joining; pRb, retinoblastoma protein; siRNA, small interfering RNA; DDR, DNA damage response Received 14.8.09; revised 26.10.09; accepted 10.11.09; Edited by N Bazan; published online 29.1.10 Cell-cycle entry is required for NHEJ activation A Tomashevski et al 1190 ends. Ku facilitates the recruitment of Artemis-DNA–protein- repairable DNA damage generated by hydrogen peroxide kinase catalytic subunit (PKcs) complex, which processes (H2O2). H2O2, a product of normal oxygen metabolism, is the ends to prepare them for ligation.17 extensively used to induce oxidative stress in cell culture cell In this study, we found that activation of NHEJ in postmitotic models and is known to induce DSBs.12,16 Cultures of neurons was accompanied by phosphorylation of pRb at cortical neurons, according to our previous results, yielded S807/811 directed by cyclin-C-associated kinase activity, B99% pure neuronal populations. The purity of neuronal which was previously shown to be sufficient for G0-G1 populations was controlled by the expression of the specific transition.14 The abrogation of cell-cycle entry compromised neuronal marker, NeuN (data not shown). Previously, we NHEJ repair, while forcing G1 entry caused NHEJ activation demonstrated that 5 mMH2O2 induces repairable DSBs and even in the absence of DNA lesions. Together, these results does not induce apoptosis in postmitotic cortical neurons.12 suggest the need for resting cells to re-enter the cell cycle to We used phosphorylated H2AX (gH2AX) as a marker of activate the NHEJ repair machinery. DSB formation.12 The kinetics of gH2AX accumulation demonstrates the repairable character of DSBs induced by 5 mMH2O2, as illustrated by a significant increase in gH2AX Results expression compared with that in untreated neurons, followed by its significant reduction (Figure 1a). The 5 lMH2O2 generates repairable DSBs in postmitotic analysis of apoptotic markers, including cleavage of neurons. To investigate the effect of non-lethal DSBs on caspase-3 (Figure 1b) and examination of apoptotic neuron neurons, we exposed postmitotic rat cortical neurons to nuclei (data not shown), revealed that by 24 h after exposure a Hours after H2O2 b 0.5 1 2 4 6 12 24 NC PC 24 h NC PC Cleaved γ H2AX caspase-3 β-tubulin β-tubulin 2 * 1.6 ** c ppRb S807/S811 pRb H2AX 1.2 γ E2F1 0.8 expression cycC Relative 0.4 CDK3 0 β-tubulin 0.512461224NCPC 0.5 1 2 4 6 12 24 NC PC Hours after H2O2 Hours after H2O2 1.2 d IPK:cyc C * ** ppRb S807/S811 IP:cyc C 1 ** E2F1 cycC 0.8 IPK:cyc D1 CDK3 0.6 IP:cyc D1 0.4 IPK:cyc E Expression Relative Protein 0.2 IP:cyc E 0.5 1 2 4 24 NC PC IgG 0 0.512461224NCPC Hours after H2O2 Hours after H2O2 e IP blot:: cycC blot:: CDK3 1 h NC IgG Figure 1 DSB induction is associated with the cell-cycle entry of postmitotic cortical neurons. (a, b) Immunoblot analysis of gH2AX and apoptotic caspase-3 cleavage in neurons treated with 5 mMH2O2. Negative control (NC): untreated cultures; positive control (PC): extract from staurosporine-treated Jurkat cells. The values are the means and S.D. (n ¼ 5); *Po0.002; **Po0.001. (c) Immunoblot analysis of the expression of cell-cycle-related proteins in neurons exposed to 5 mMH2O2. Negative control (NC): untreated neurons; positive control (PC): extracts from proliferating HeLa cells. The values are the means and S.D. (n ¼ 4); *Po0.002; **Po0.001 compared with corresponding NC. (d) Lysates from cortical neurons were prepared at the indicated times and subjected to immune precipitation (IP) using anti-cyclin-C, anti-cyclin-D1 and anti-cyclin-E. Precipitates were tested for in vitro kinase activity using Rb-769 as substrate (IPK). Negative control (NC): untreated neurons; positive control (PC): proliferating HeLa cells. Isospecific control (IgG): normal rabbit or mouse IgG. (e) Lysates from cortical neurons treated with 5 mMH2O2 were subjected to immunoprecipitation followed by immunoblot analysis. Anti-cyclin-C precipitates were analyzed using anti-CDK3 antibody and anti-CDK3 precipitates were analyzed using anti-cyclin-C antibody. Normal rabbit IgG (IgG) was used as control for both anti-CDK3 and cyclin-C antibodies Cell Death and Differentiation Cell-cycle entry is required for NHEJ activation A Tomashevski et al 1191 to 5 mMH2O2, the neuronal cultures did not undergo previously observed pRb phosphorylation at S807/811 in 14,18 apoptosis, consistent with our data on gH2AX expression.